Urinary Trimethylamine N-oxide Research Articles

#1801 Gut dysbiosis contributes to TMAO accumulation in CKD

Abstract Background and Aims Chronic kidney disease (CKD) patients face a substantial morbidity risk, especially related to cardiovascular complications. The imbalance of circulating metabolites influences inter-organ crosstalk and disease pathophysiology. In this regard, metabolites of microbial origin have gained increasing attention. Trimethylamine N-oxide (TMAO) originates from microbial Trimethylamine (TMA) metabolism and is found at high concentrations in the blood of CKD patients. A high TMAO concentration is known to be an independent risk factor for cardiovascular disease. This study aims to investigate the extent to which high concentrations of TMAO in the blood are due to increased bacterial production or are mainly a consequence of impaired renal clearance. Methods We used existing biomaterials and clinical data of a pediatric CKD cohort, consisting of patients enrolled at CKD stage G3-5, including G5 patients treated with hemodialysis (HD), as well as patients after kidney transplantation (KT) and individuals with normal kidney function. We analyzed TMAO in plasma and urine samples using LC-MS/MS and developed a novel gene targeting assay (GTA) that quantifies the abundance of the bacterial target genes (CutC, CntA) encoding for the key enzymes of microbial TMA synthesis in each fecal sample. Results Plasma TMAO levels were stage-dependently elevated in CKD, with HD patients showing the highest levels (75 ± 46 µmol/l). While urinary TMAO levels were lower in the CKD groups, TMAO/creatinine ratios and fractional excretion were not significantly different in CKD compared to controls. Fecal microbial gene abundance of both CutC and CntA genes was elevated across CKD stages and correlated with both plasma TMAO levels and eGFR. Although KT patients exhibited an eGFR range comparable to controls, they still showed higher plasma TMAO levels and elevated TMAO-associated gene abundances. In a regression analysis, this gene abundance was associated with plasma TMAO independent of eGFR in a model adjusted for age, sex, and BMI. Using a likelihood ratio test, we showed that adding fecal microbial gene abundance to the model provided a significantly better fit compared to only eGFR, age, sex, and BMI. Conclusion Our data indicate that the production of TMA by intestinal bacteria contributes significantly to the accumulation of TMAO in patients with CKD and after kidney transplantation. This finding opens the possibility for new potential interventions in the management of patients with CKD.

Exposure Patterns of Multiple Pesticides and Urinary Alterations of Trimethylamine (TMA) and Trimethylamine N-Oxide (TMAO) among Pregnant Women.

Environmental exposure to pesticides is widespread and commonly occurs in mixtures, and pregnant women are particularly concerned. Trimethylamine N-oxide (TMAO), a dietary-derived catabolite linked to cardiometabolic disease, may be influenced by pesticide exposure. However, limited knowledge exists regarding the associations, and studies have primarily been restricted to single pesticides. To address this gap, we examined the associations of pesticide exposure with urinary TMA, TMAO, and TMAO-to-TMA ratio among 1067 pregnant women residing in a rural area. Latent class analysis (LCA) was applied to identify distinct patterns of pesticide exposure based on urinary metabolite concentrations. Multiple linear regression (MLR) models and Bayesian kernel machine regression (BKMR) models were employed to estimate the effects of single pesticides, exposure patterns, and combined exposure. Four distinct pesticide exposure patterns were identified. Higher concentrations of pesticide metabolites were found to be associated with increased urinary TMAO levels (β: 0.043 to 0.254, p < 0.05). In the MLR models for individual pesticides and exposure patterns, carbofuran phenol (CFP), pentachlorophenol (PCP), and 2-phenylphenol (OPP) exhibited positive associations with urinary TMA concentrations, while pyrethroid pesticide metabolites were correlated with the up-regulation of the TMAO-to-TMA ratio. Significant positive associations of pesticide mixtures with TMA, TMAO, and TMAO-to-TMA ratio were observed in the BKMR model, primarily driven by PCP and trans-DCCA. Our findings provided a perspective to reveal that the mechanisms and contributions to TMAO alterations may vary among different pesticides in mixture exposure.

A18 INTERKINGDOM DYNAMICS AND NUTRITION ARE ASSOCIATED WITH DIVERGENT MATURATIONAL PATTERNS OF THE INFANT BACTERIAL AND FUNGAL GUT MICROBIOME

Abstract Background Early life has been identified as a critical window, during which time deviations from typical patterns of gut microbiome maturation have been associated with adverse health outcomes later in life. In the first 2-3 years of life, the infant gut microbiome undergoes ecological shifts characterized by increasing bacterial alpha diversity and variable changes in fungal alpha diversity. Research has shown not all infants follow these maturational trends, but our understandings of the factors linked to atypical microbiome maturation patterns are limited. Purpose We assessed bacterial and fungal gut microbiome maturation in early life to determine if atypical maturational patterns were observed in otherwise healthy infants and identify factors associated with these patterns. Method In 100 infants from the CHILD Cohort Study, we assessed the bacterial and fungal gut microbiome in stool samples collected at 3 and 12 months of age using 16S and ITS2 Illumina sequencing, respectively. We performed untargeted metabolomics on urine samples collected at 3 and 12 months using liquid chromatography-mass spectrometry/mass spectrometry. Microbiome and metabolomic measures were evaluated by ecological and multivariate analyses using RStudio. Result(s) Gut microbiome analyses revealed 24% and 20% of infants displayed atypical alpha diversity trajectories in the first year of life for bacteria or fungi, respectively. Atypical patterns were linked to reduced abundance of Bacteroides and increased Candida at 3 months. Functional analysis revealed an atypical bacterial alpha diversity trend was associated with elevated urinary trimethylamine N-oxide, creatine, indole acetic acid, and 2-furoylglycine, and an atypical fungal trend was associated with elevated urinary lactate. Using decision trees, the strongest predictors of atypical alpha diversity trends were interkingdom dynamics, breastfeeding duration, and maternal diet during pregnancy. Logistic regression revealed an atypical bacterial trend was positively associated with delivery via C-section and inversely associated with exclusive breastfeeding at 3 months, and an atypical fungal trend was positively associated with gestational consumption of artificially sweetened beverages and inversely associated with prenatal antibiotics. Interkingdom network analyses revealed the gut microbiome of infants with an atypical bacterial or fungal alpha diversity trend displayed a greater number of interkingdom interactions reflective of a less stable or immature gut microbiome. Conclusion(s) Our findings reveal a substantial proportion of infants display atypical patterns of gut microbiome maturation in the first year of life. While known microbiome-modifying factors were important determinants of maturational patterns, these factors were generally less influential than interkingdom influences. Together, this highlights the importance of interkingdom analyses at the individual level to generate more nuanced understandings of maturational trajectories in early life. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; Alberta Children's Hospital Research Institute Disclosure of Interest None Declared

A family study of compound variants of flavin-containing monooxygenase 3 (FMO3) in Japanese subjects found by urinary phenotyping for trimethylaminuria

Phenotype–gene analyses and the increasing availability of mega-databases have revealed the impaired human flavin-containing monooxygenase 3 (FMO3) variants associated with the metabolic disorder trimethylaminuria. In this study, a novel compound variant of FMO3, p.[(Val58Ile; Tyr229His)], was identified in a 1-year-old Japanese girl who had impaired FMO3 metabolic capacity (70%) in terms of urinary trimethylamine N-oxide excretion levels divided by total levels of trimethylamine and its N-oxide. One cousin in the family had the same p.[(Val58Ile); (Tyr229His)]; [(Glu158Lys; Glu308Gly)] FMO3 haplotype and had a similar FMO3 metabolic capacity (69%). In a family study, the novel p.[(Val58Ile); (Tyr229His)] compound FMO3 variant was also detected in the proband 1's mother and aunt. Another novel compound FMO3 variant p.[(Glu158Lys; Met260Lys; Glu308Gly; Ile426Thr)] was identified in a 7-year-old girl, proband 2. This novel compound FMO3 variant was inherited from her mother. Recombinant FMO3 Val58Ile; Tyr229His variant and Glu158Lys; Met260Lys; Glu308Gly; Ile426Thr variant showed moderately decreased capacities for trimethylamine N-oxygenation compared to wild-type FMO3. Analysis of trimethylaminuria phenotypes in family studies has revealed compound missense FMO3 variants that impair FMO3-mediated N-oxygenation in Japanese subjects; moreover, these variants could result in modified drug clearances.

Monosodium glutamate consumption reduces the renal excretion of trimethylamine N-oxide and the abundance of Akkermansia muciniphila in the gut

We previously demonstrated that monosodium glutamate (MSG) consumption increases trimethylamine (TMA) level in the renal tissue as well as dimethylamine and methylamine levels in urine of rats, suggesting the effects of MSG on humans. To better define the findings, we investigated whether MSG consumption alters serum trimethylamine N-oxide (TMAO) level, and as a consequence, induces kidney injury in the rat model. Adult male Wistar rats (n = 40) were randomized to be fed with a standard diet (control group) or a standard diet with 0.5, 1.5 or 3.0 g% MSG corresponding to 7, 21, or 42 g/day in 60 kg man, respectively in drinking water (MSG-treated groups), or a standard diet with 3.0 g% MSG in drinking water which was withdrawn after 4 weeks (MSG-withdrawal group). Blood and urine samples were collected to analyze the TMAO levels using 1H NMR and markers of kidney injury. Fecal samples were also collected for gut microbiota analysis. We found serum TMAO levels increased and urinary TMAO excretion decreased during MSG consumption, in parallel with the increase of the neutrophil gelatinase-associated lipocalin (NGAL) excretion which subsided with the withdrawal of MSG. The fecal 16 S rRNA analysis during MSG consumption showed gut microbiota changes with a consistent suppression of Akkermansia muciniphila, a mucin producing bacteria, but not of TMA-producing bacteria. In conclusions, our findings suggested that prolonged high dose MSG consumption may cause TMAO accumulation in the blood via reduction of renal excretion associated with acute kidney injury. The mechanisms by which MSG reduced TMAO excretion require further investigation.

Associations of Diet with Urinary Trimethylamine-N-Oxide (TMAO) and Its Precursors among Free-Living 10-Year-Old Children: Data from SMBCS.

Trimethylamine-N-oxide (TMAO), a diet-derived cometabolite linked to cardiometabolic disease, has been associated with elevated dietary status, particularly in people with kidney failure and adults with dietary modulations. However, the influence of the current diet on TMAO levels in free-living children has not been adequately described. This study was to explore associations of food compositions and dietary diversity with urinary TMAO and its precursor concentrations. Urinary TMAO and its precursor concentrations of 474 healthy children from the Sheyang Mini Birth Cohort were quantified by ultra-performance liquid chromatography–Q Exactive high-resolution mass spectrometer (UPLC-Q Exactive HRMS). Individual food compositions from 24 h dietary recall data were classified into 20 groups and diversity scores were calculated according to the guidelines of the Food and Agriculture Organization of the United Nations (FAO). Associations of urinary TMAO and its precursors with food compositions and dietary diversity scores were assessed by generalized linear regression models. In models adjusted for potential confounders, urinary TMAO was significantly associated with intakes of fish (β, regression coefficient = 0.155, p < 0.05) and vegetables (β = 0.120, p < 0.05). Eggs intake showed positive associations with TMAO’s precursors (trimethylamine: β = 0.179, p < 0.05; choline: β = 0.181, p < 0.05). No association between meat intake and TMAO was observed, whereas meat and poultry intakes were related to the levels of acetyl-L-carnitine and L-carnitine (β: 0.134 to 0.293, p < 0.05). The indicators of dietary diversity were positively correlated to TMAO concentration (β: 0.027 to 0.091, p < 0.05). In this free-living children-based study, dietary factors were related to urinary TMAO and its precursors, especially fish, meat, and eggs. As such, dietary diversity was positively related to the level of TMAO.

The Influence of Animal- or Plant-Based Diets on Blood and Urine Trimethylamine-N-Oxide (TMAO) Levels in Humans.

The aim of the review was to evaluate which diets are associated with higher TMAO levels. Several studies have shown that plasma and urinary levels of trimethylamine N-oxide (TMAO) are a reliableindicator of cardiovascular disease risk. Diet certainly has a strong influence on TMAO levels, but there is stilluncertainty about which diet is the most effective in reducing this risk factor. PubMed, Web of Science and Scopus were searched for studies that were published up until July 1, 2021 usingspecific keywords. In total, 447 studies were evaluated, of which papers on individual foods or supplements, orconducted in children, in vitro or in animal model studies were excluded. Twenty-five studies were included in thisreview. Three studies showed that caloric restriction and (visceral) weight loss improve TMAO levels. Six out ofeight studies revealed beneficial effects of plant-based diets on plasma or urinary TMAO concentrations. Most ofthe studies demonstrated that a diet high in protein, particularly of animal origin, such as diets rich in fish or redmeat, have negative effects on TMAO levels. Most studies that have evaluated the relationship between diet andplasma or urinary concentrations of TMAO seem to indicate that plant-based diets (Mediterranean, vegetarian andvegan) are effective in improving TMAO levels, while animal-based diets appear to have the opposite effect.Further long-term studies are needed to assess whether vegetarian or vegan diets are more effective than theMediterranean diet in reducing TMAO levels.

Metabolic Alterations Identified in Urine, Plasma and Aortic Smooth Muscle Cells Reflect Cardiovascular Risk in Patients with Programmed Coronary Artery Bypass Grafting.

Atherosclerosis is the predominant pathology associated to premature deaths due to cardiovascular disease. However, early intervention based on a personalized diagnosis of cardiovascular risk is very limited. We have previously identified metabolic alterations during atherosclerosis development in a rabbit model and in subjects suffering from an acute coronary syndrome. Here we aim to identify specific metabolic signatures which may set the basis for novel tools aiding cardiovascular risk diagnosis in clinical practice. In a cohort of subjects with programmed coronary artery bypass grafting (CABG), we have performed liquid chromatography and targeted mass spectrometry analysis in urine and plasma. The role of vascular smooth muscle cells from human aorta (HA-VSMCs) was also investigated by analyzing the intra and extracellular metabolites in response to a pro-atherosclerotic stimulus. Statistically significant variation was considered if p value < 0.05 (Mann-Whitney test). Urinary trimethylamine N-oxide (TMAO), arabitol and spermidine showed higher levels in the CVrisk group compared with a control group; while glutamine and pantothenate showed lower levels. The same trend was found for plasma TMAO and glutamine. Plasma choline, acetylcholine and valine were also decreased in CVrisk group, while pyruvate was found increased. In the secretome of HA-VSMCs, TMAO, pantothenate, glycerophosphocholine, glutathion, spermidine and acetylcholine increased after pro-atherosclerotic stimulus, while secreted glutamine decreased. At intracellular level, TMAO, pantothenate and glycerophosphocholine increased with stimulation. Observed metabolic deregulations pointed to an inflammatory response together with a deregulation of oxidative stress counteraction.

Association of Urinary and Plasma Levels of Trimethylamine N-Oxide (TMAO) with Foods.

Introduction: Trimethylamine N-oxide (TMAO) may play a key mediator role in the relationship between the diet, gut microbiota and cardiovascular diseases, particularly in people with kidney failure. The aim of this review is to evaluate which foods have a greater influence on blood or urinary trimethylamine N-oxide (TMAO) levels. Methods: 391 language articles were screened, and 27 were analysed and summarized for this review, using the keywords “TMAO” AND “egg” OR “meat” OR “fish” OR “dairy” OR “vegetables” OR “fruit” OR “food” in December 2020. Results: A strong correlation between TMAO and fish consumption, mainly saltwater fish and shellfish, but not freshwater fish, has been demonstrated. Associations of the consumption of eggs, dairy and meat with TMAO are less clear and may depend on other factors such as microbiota or cooking methods. Plant-based foods do not seem to influence TMAO but have been less investigated. Discussion: Consumption of saltwater fish, dark meat fish and shellfish seems to be associated with an increase in urine or plasma TMAO values. Further studies are needed to understand the relationship between increased risk of cardiovascular disease and plasma levels of TMAO due to fish consumption. Interventions coupled with long-term dietary patterns targeting the gut microbiota seem promising.

Effect of fructooligosaccharide on endothelial function in CKD patients: a randomized controlled trial.

Microbiota-derived uremic toxins have been associated with inflammation that could corroborate with endothelial dysfunction (ED) and increase cardiovascular risk in patients with chronic kidney disease (CKD). This trial aimed to evaluate the effect of the prebiotic fructooligosaccharide (FOS) on endothelial function and arterial stiffness in nondialysis CKD patients. In a double-blind controlled trial, 46 nondiabetic CKD patients were randomized to receive 12 g/day of FOS or placebo (maltodextrin) for 3 months. Total p-cresyl sulfate (PCS) and indoxyl sulfate by high-performance liquid chromatography, urinary trimethylamine N-oxide by mass spectrometry, C-reactive protein, interleukin-6 (IL-6), serum nitric oxide and stroma-derived factor-1 alfa were measured at baseline and at the end of follow-up; endothelial function was assessed through flow-mediated dilatation (FMD) and arterial stiffness by pulse wave velocity (PWV). The mean (± standard deviation) age of the study participants was 57.6 ± 14.4 years, with an estimated glomerular filtration rate of 21.3 ± 7.3 mL/min/1.73 m2. During the follow-up, regarding the inflammatory markers and uremic toxins, there was a significant decrease in IL-6 levels (3.4 ± 2.1 pg/mL versus 2.6 ± 1.4 pg/mL; P = 0.04) and a trend toward PCS reduction (55.4 ± 38.1 mg/L versus 43.1 ± 32.4 mg/L, P = 0.07) only in the prebiotic group. Comparing both groups, there was no difference in FMD and PWV. In an exploratory analysis, including a less severe ED group of patients (FMD ≥2.2% at baseline), FMD remained stable in the prebiotic group, while it decreased in the placebo group (group effect P = 0.135; time effect P = 0.012; interaction P = 0.002). The prebiotic FOS lowered circulating levels of IL-6 in CKD patients and preserved endothelial function only in those with less damaged endothelium. No effect of FOS in arterial stiffness was observed.

Chlorogenic acid inhibits trimethylamine-N-oxide formation and remodels intestinal microbiota to alleviate liver dysfunction in high L-carnitine feeding mice.

High L-carnitine ingestion has been shown to cause liver injury, mechanically due to an elevated circulating level of trimethylamine-N-oxide (TMAO), a gut microbiota-derived metabolite from L-carnitine. This study aimed to investigate whether chlorogenic acid (CGA), a health-promoting polyphenol, could inhibit TMAO formation and thereafter might prevent L-carnitine-induced liver injury in mice. Feeding of mice with 3% L-carnitine in drinking water increased the serum and urinary levels of TMAO (p < 0.01 vs. Normal), whereas the serum and urinary TMAO formation was sharply reduced by CGA administration (p < 0.01). At the phylum level, CGA inhibited the L-carnitine-induced increase in the abundance of Firmicutes and Proteobacteria, while it promoted Bacteroidetes. At the genus level, CGA notably increased the abundance of Akkermansia and Bacteroides, but reduced the population of Erysipelatoclostridium, Faecalibaculum and Erysipelotrichaceae in high L-carnitine feeding mice. Meanwhile, CGA caused strong inhibition against the increase of liver injury markers (i.e. AST, ALT and ALP), hepatic inflammatory cytokines (i.e. IL-1, IL-6, TNF-α and TNF-β) and dyslipidemia (i.e. TC, TG, LDL-C and HDL-C) in L-carnitine-fed mice (p < 0.05). These findings suggest that CGA holds great potential to alleviate liver dysfunction induced by high L-carnitine ingestion. The beneficial effect might be attributed to the protection against TMAO formation and the improvement of the health-promoting gut microbiota, as well as the antioxidant and anti-inflammatory properties of CGA.

1H NMR metabolomic and transcriptomic analyses reveal urinary metabolites as biomarker candidates in response to protein undernutrition in adult rats.

Protein undernutrition contributes to the development of various diseases in broad generations. Urinary metabolites may serve as non-invasive biomarkers of protein undernutrition; however, this requires further investigation. We aimed to identify novel urinary metabolites as biomarker candidates responsive to protein undernutrition. Adult rats were fed control (CT; 14 % casein) or isoenergetic low-protein (LP; 5 % casein) diets for 4 weeks. 1H NMR metabolomics was applied to urine, plasma and liver samples to identify metabolites responsive to protein undernutrition. Liver samples were subjected to mRNA microarray and quantitative PCR analyses to elucidate the mechanisms causing fluctuations in identified metabolites. Urinary taurine levels were significantly lower in the LP group than in the CT group at week 1 and remained constant until week 4. Hepatic taurine level and gene expression level of cysteine dioxygenase type 1 were also significantly lower in the LP group than in the CT group. Urinary trimethylamine N-oxide (TMAO) levels were significantly higher in the LP group than in the CT group at week 2 and remained constant until week 4. Hepatic TMAO level and gene expression levels of flavin-containing mono-oxygenase 1 and 5 were also significantly higher in the LP group than in the CT group. In conclusion, urinary taurine and TMAO levels substantially responded to protein undernutrition. Furthermore, changes in hepatic levels of these metabolites and gene expressions associated with their metabolic pathways were also reflected in their fluctuating urinary levels. Thus, taurine and TMAO could act as non-invasive urinary biomarker candidates to detect protein undernutrition.

Effect of Choline Forms and Gut Microbiota Composition on Trimethylamine-N-Oxide Response in Healthy Men.

Background: Trimethylamine-N-oxide (TMAO), a choline-derived gut microbiota-dependent metabolite, is a newly recognized risk marker for cardiovascular disease. We sought to determine: (1) TMAO response to meals containing free versus lipid-soluble choline and (2) effects of gut microbiome on TMAO response. Methods: In a randomized, controlled, double-blinded, crossover study, healthy men (n = 37) were provided meals containing 600 mg choline either as choline bitartrate or phosphatidylcholine, or no choline control. Results: Choline bitartrate yielded three-times greater plasma TMAO AUC (p = 0.01) and 2.5-times greater urinary TMAO change from baseline (p = 0.01) compared to no choline and phosphatidylcholine. Gut microbiota composition differed (permutational multivariate analysis of variance, PERMANOVA; p = 0.01) between high-TMAO producers (with ≥40% increase in urinary TMAO response to choline bitartrate) and low-TMAO producers (with <40% increase in TMAO response). High-TMAO producers had more abundant lineages of Clostridium from Ruminococcaceae and Lachnospiraceae compared to low-TMAO producers (analysis of composition of microbiomes, ANCOM; p < 0.05). Conclusion: Given that phosphatidylcholine is the major form of choline in food, the absence of TMAO elevation with phosphatidylcholine counters arguments that phosphatidylcholine should be avoided due to TMAO-producing characteristics. Further, development of individualized dietary recommendations based on the gut microbiome may be effective in reducing disease risk

In older women, a high-protein diet including animal-sourced foods did not impact serum levels and urinary excretion of trimethylamine-N-oxide

Diets including red meat and other animal-sourced foods may increase proteolytic fermentation and microbial-generated trimethylamine (TMA) and, subsequently, trimethylamine-N-oxide (TMAO), a metabolite associated with increased risk of cardiovascular disease and dementia. It was hypothesized that compared to usual dietary intake, a maintenance-energy high-protein diet (HPD) would increase products of proteolytic fermentation, whereas adjunctive prebiotic, probiotic, and synbiotic supplementation may mitigate these effects. An exploratory aim was to determine the association of the relative abundance of the TMA-generating taxon, Emergencia timonensis, with serum and urinary TMAO. At 5 time points (usual dietary intake, HPD diet, HPD + prebiotic, HPD + probiotic, and HPD + synbiotic), urinary (24-hour) and serum metabolites and fecal microbiota profile of healthy older women (n = 20) were measured by liquid chromatography–tandem mass spectrometry and 16S rRNA gene amplicon sequencing analyses, respectively. The HPD induced increases in serum levels of l-carnitine, indoxyl sulfate, and phenylacetylglutamine but not TMAO or p-cresyl sulfate. Urinary excretion of l-carnitine, indoxyl sulfate, phenylacetylglutamine, and TMA increased with the HPD but not TMAO or p-cresyl sulfate. Most participants had undetectable levels of E.timonensis at baseline and only 50% during the HPD interventions, suggesting other taxa are responsible for the microbial generation of TMA in these individuals. An HPD diet with or without a prebiotic, probiotic, or synbiotic elicited an increase in products of proteolytic fermentation. The urinary l-carnitine response suggests that the additional dietary l-carnitine provided was primarily bioavailable, providing little substrate for microbial conversion to TMA and subsequent TMAO formation.

Background Diet Influences TMAO Concentrations Associated with Red Meat Intake without Influencing Apparent Hepatic TMAO-Related Activity in a Porcine Model.

Red meat has been associated with an increased cardiovascular disease (CVD) risk, possibly through gut microbial-derived trimethylamine-N-oxide (TMAO). However, previous reports are conflicting, and influences from the background diet may modulate the impact of meat consumption. This study investigated the effect of red and white meat intake combined with two different background diets on urinary TMAO concentration and its association with the colon microbiome in addition to apparent hepatic TMAO-related activity. For 4 weeks, 32 pigs were fed chicken or red and processed meat combined with a prudent or western background diet. 1H NMR-based metabolomics analysis was conducted on urine samples and hepatic Mrna expression of TMAO-related genes determined. Lower urinary TMAO concentrations were observed after intake of red and processed meat when consumed with a prudent compared to a western background diet. In addition, correlation analyses between urinary TMAO concentrations and relative abundance of colon bacterial groups suggested an association between TMAO and specific bacterial taxa. Diet did not affect the hepatic Mrna expression of genes related to TMAO formation. The results suggest that meat-induced TMAO formation is regulated by mechanisms other than alterations at the hepatic gene expression level, possibly involving modulations of the gut microbiota.

The association of fish consumption and its urinary metabolites with cardiovascular risk factors: the International Study of Macro-/Micronutrients and Blood Pressure (INTERMAP)

ABSTRACTBackgroundResults from observational studies regarding associations between fish (including shellfish) intake and cardiovascular disease risk factors, including blood pressure (BP) and BMI, are inconsistent.ObjectiveTo investigate associations of fish consumption and associated urinary metabolites with BP and BMI in free-living populations.MethodsWe used cross-sectional data from the International Study of Macro-/Micronutrients and Blood Pressure (INTERMAP), including 4680 men and women (40–59 y) from Japan, China, the United Kingdom, and United States. Dietary intakes were assessed by four 24-h dietary recalls and BP from 8 measurements. Urinary metabolites (2 timed 24-h urinary samples) associated with fish intake acquired from NMR spectroscopy were identified. Linear models were used to estimate BP and BMI differences across categories of intake and per 2 SD higher intake of fish and its biomarkers.ResultsNo significant associations were observed between fish intake and BP. There was a direct association with fish intake and BMI in the Japanese population sample (P trend = 0.03; fully adjusted model). In Japan, trimethylamine-N-oxide (TMAO) and taurine, respectively, demonstrated area under the receiver operating characteristic curve (AUC) values of 0.81 and 0.78 in discriminating high against low fish intake, whereas homarine (a metabolite found in shellfish muscle) demonstrated an AUC of 0.80 for high/nonshellfish intake. Direct associations were observed between urinary TMAO and BMI for all regions except Japan (P < 0.0001) and in Western populations between TMAO and BP (diastolic blood pressure: mean difference 1.28; 95% CI: 0.55, 2.02 mmHg; P = 0.0006, systolic blood pressure: mean difference 1.67; 95% CI: 0.60, 2.73 mmHg; P = 0.002).ConclusionsUrinary TMAO showed a stronger association with fish intake in the Japanese compared with the Western population sample. Urinary TMAO was directly associated with BP in the Western but not the Japanese population sample. Associations between fish intake and its biomarkers and downstream associations with BP/BMI appear to be context specific. INTERMAP is registered at www.clinicaltrials.gov as NCT00005271.

The Relationship between Fish Intake and Urinary Trimethylamine-N-Oxide.

Fish intake is reported to be associated with certain health benefits; however, accurate assessment of fish intake is still problematic. The objective of this study is to identify fish intake biomarkers and examine relationships with health parameters in a free-living population. In the NutriTech study, ten participants randomized into the fish group consume increasing quantities of fish for 3 days per week for 3 weeks. Urine is analyzed by NMR spectroscopy. Trimethylamine-N-oxide (TMAO), dimethylamine, and dimethyl sulfone are identified and display significant dose-response with intake (p<0.05). Fish consumption yields a greater increase in urinary TMAO compared to red meat. Biomarker-derived fish intake is calculated in the National Adult Nutrition Survey cross-sectional study. However, the correlation between fish intake and TMAO (r=0.148, p<0.01) and that between fish intake and calculated fish intake (r=0.142, p<0.01) are poor. In addition, TMAO shows significantly positive correlation with serum insulin and insulin resistance in males and the relationship is more pronounced for males with high dietary fat intake. Urinary TMAO displays a strong dose-response relationship with fish intake; however, use of TMAO alone is insufficient to determine fish intake in a free-living population.

Urinary TMAO Levels Are Associated with the Taxonomic Composition of the Gut Microbiota and with the Choline TMA-Lyase Gene (cutC) Harbored by Enterobacteriaceae.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

Decaisnea insignis Seed Oil Inhibits Trimethylamine-N-oxide Formation and Remodels Intestinal Microbiota to Alleviate Liver Dysfunction in l-Carnitine Feeding Mice.

Elevated circulating level of the intestinal microbiota-derived l-carnitine metabolite trimethylamine-N-oxide (TMAO) has recently been linked to many chronic diseases. The purpose of our study was to investigate the effects of omega-7-enriched Decaisnea insignis seed oil (DISO) on reducing TMAO formation to prevent the l-carnitine-induced hepatic damage in mice. Feeding of mice with 3% l-carnitine in drinking water clearly increased the serum and urinary levels of TMAO (p < 0.05 vs Normal), whereas the serum and urinary TMAO formation was sharply reduced by DISO administration (p < 0.05). Meanwhile, DISO resulted in strong inhibition against the elevation of hepatic injury marker (AST, ALT, and ALP) activities and dyslipidemia (TC, TG, LDL-C, and HDL-C), as well as liver inflammatory cytokine (IL-1, IL-6, TNF-α, and TNF-β) release in l-carnitine-fed mice (p < 0.05). As revealed by 16S rDNA gene sequencing, DISO significantly inhibited the l-carnitine-induced elevations in the abundance of Firmicutes, Proteobacteria, and Erysipelotrichaceae and the increases in the proportion of Lactobacillus and Akkermansia, revealing that DISO attenuated the l-carnitine-caused gut dysbiosis. These findings suggested that DISO could alleviate liver dysfunction in l-carnitine-fed mice, which might be due to the protection against TMAO formation by modulating the gut microbiota.

The interplay between microbiota‐dependent metabolite trimethylamine N‐oxide, Transforming growth factor β/SMAD signaling and inflammasome activation in chronic kidney disease patients: A new mechanistic perspective

Chronic kidney disease (CKD) signifies a frequently life-threatening condition influencing kidney structure and function. Despite its irrefutable importance, its exact pathogenesis is not completely clarified. However, CKD is known to be associated with accumulated uremic toxins/metabolites, interstitial fibrosis, and systemic inflammation. So we aimed to investigate the role of microbiota-dependent metabolite trimethylamine N-oxide (TMAO), transforming growth factor β (TGFβ)/SMAD signaling, and inflammasome activation in CKD pathogenesis through its different stages. Eighty patients with CKD of stages 2 to 4 in addition 15 healthy control subjects were enrolled. SMAD3 and nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) messenger RNA (mRNA) expressions from whole blood were assessed by quantitative real-time polymerase chain reaction (RT-PCR). Serum TGF-β1 and interleukin-1β (IL-1β) levels were estimated by the enzyme-linked immunosorbent assay. Plasma and urinary TMAO levels were measured. Oxidative stress markers were also assessed. SMAD3 and NLRP3 mRNA expressions were significantly upregulated in patients with CKD. Likewise, serum TGF-β1 and IL-1β levels were significantly elevated in patients with CKD, with increase in plasma and urinary TMAO levels and altered redox status throughout different CKD stages. The study documented that TMAO could be used as a reliable biomarker to evaluate CKD progression; being linked to TGF-β/SMAD signaling, NLRP3 inflammasome activation as well as being a noninvasive applicable technique.